CN115785266A - Antibody for resisting eggs of ancient elephant pests and application thereof - Google Patents

Abstract

The application provides an antibody for resisting eggs of ancient elephant (Sitophilus) pests, a test strip for detecting the eggs of the ancient elephant pests, a preparation method and a use method of the test strip, and a kit containing the antibody or the test strip.

Description

Antibody for resisting eggs of ancient elephant pests and application thereof

Technical Field

The application relates to the fields of molecular biology and immunology, in particular to an antibody for resisting eggs of ancient elephant (Sitophilus) pests, a test strip for detecting eggs of ancient elephant pests, a preparation method and a use method thereof, and a kit containing the antibody or the test strip.

Background

Zeamais (s. Zeamais) is one of the major grain storage pests. In flour processing plants and rice processing plants, the pests can cause great grain pollution. Corn is a decaying grain storage pest, and can directly take grain and lay worm eggs in the grain, and the process from the development of the worm eggs to adults is completed in the grain. After the adult develops, the adult will drill out from the grains eaten by the moth, mate and eat other grains.

The main research and application directions today are how to effectively trap adults in cereals, and how to use effective insecticides to kill adults or to inhibit the hatching of worm eggs and larvae. In practical application, the effect of trapping adults on controlling the stored grain insects is not obvious, the fumigation treatment of the pesticide is relatively direct, but the use of the pesticide relates to the problem of food safety, and the practical application is not easy to operate due to the problems of corrosion of chemical substances to warehouses, permeability of the chemical substances in grain particles and the like. In the field of grain research nowadays, the main research direction is to find natural pesticide to effectively repel or kill stored grain pests and their eggs ^{[1][2][3][4][5]} However, the complexity and cost of natural insecticides have resulted in their being completely unacceptable at the current industrial level.

Thus, there is still a need to discover and effectively eliminate tacrolimus from grains early on.

Disclosure of Invention

The application obtains specific egg protein in natural eggs based on protein analysis of the eggs of the elephant corn insects, obtains a hybridoma cell strain WMAZC2 capable of secreting specific recognition of the egg protein antibody of the elephant corn insects by immunizing a mouse based on an immunological method, has the preservation number of CGMCC 19184, can specifically recognize the egg protein of the elephant corn insects by a secreted monoclonal antibody, can be used for preparing a test strip for specifically detecting the egg protein, can sensitively detect the eggs of the elephant corn insects in grain products such as rice, wheat and the like, and achieves the purpose of early warning of insect pest outbreak.

Specifically, the application provides the following technical scheme:

in a first aspect, the present application provides an antibody or antigen-binding fragment thereof that is capable of specifically binding to egg proteins of an ancient elephant pest egg.

In some embodiments, the antibody is encoded by a accession number of CGMCC NO:19184. the hybridoma cell of (3).

In a second aspect, the present application provides a monoclonal antibody encoded by a nucleic acid sequence having a accession number of CGMCC NO:19184.

In a third aspect, the present application provides a hybridoma cell having a accession number of CGMCC NO:19184.

in a fourth aspect, the present application provides a test strip for detecting eggs of an ancient elephant pest, comprising the antibody of the first aspect or the monoclonal antibody of the second aspect.

In a fifth aspect, the present application provides a kit for detecting eggs of an ancient elephant pest, comprising the antibody of the first aspect, the monoclonal antibody of the second aspect, or the test strip of the fourth aspect.

In a sixth aspect, the application provides a method for detecting eggs of ancient elephant pests, which comprises contacting the antibody of the first aspect, the monoclonal antibody of the second aspect, the reagent of the test strip of the fourth aspect or the kit of the fifth aspect with a sample to be detected,

if the antibody binds to the sample, it indicates the presence of eggs from an ancient insect pest in the sample.

In a seventh aspect, the application provides use of the antibody of the first aspect, the monoclonal antibody of the second aspect, the test strip of the fourth aspect, or the kit of the fifth aspect, for detecting the presence of eggs of an archaeological pest in a grain.

In any of the preceding aspects, the archaeological pest is selected from the group consisting of hordeolum zeamais, elephant archaeology, and hordeolum micranthum.

The existing method for detecting the eggs of the insects in the cereals mainly adopts a potassium permanganate dyeing method (1% (w/v) potassium permanganate solution), needs to prepare special dyeing reagents on site, needs to observe single rice one by one under a microscope, has low accuracy and long required time, and cannot meet the requirement of quickly detecting and simply identifying whether the eggs exist. The hybridoma cell strain capable of secreting the specificity recognition corn elephant egg protein antibody is obtained, the secreted monoclonal antibody can be used for preparing a test strip for specifically detecting the egg protein, the corn elephant egg in grain products such as rice and wheat can be detected very sensitively, the sensitivity is high, the operation is simple, equipment such as a dyeing or microscope is not needed, and the hybridoma cell strain is very suitable for on-site quick detection. The early warning of rice insect pests in grain storage units, grain warehouses and large rice processing factories is well assisted.

Brief description of the drawings

FIG. 1 shows the result of SDS-PAGE electrophoresis of egg proteins of weevils of corn.

FIG. 2 shows the results of the test strip of the present application for detecting eggs of a Pectenophora zeae.

Detailed description of the embodiments

The practice of the present invention will employ, unless otherwise indicated, molecular biology, microbiology, cell biology, biochemistry and immunology techniques which are conventional in the art.

Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.

This application has designed the thinking that detects cereal worm egg content to the problem of control grain insect pest, instructs the early insect pest infection condition of grain through how much of worm egg content to in time make the measure of answering, prevent extensive outbreak. For example, after egg detection is carried out on a batch of grains, the possibility of insect pest outbreak and the safe storage time before outbreak are estimated, then, for grains with serious infection, the grains are subjected to treatment such as key fumigation, dry heat baking, freezing and insect killing and the like by a factory, and the final logistics transportation condition is adjusted and the market positioning is carried out again by functional departments in the selling link, so that the phenomenon of large-scale insect growth does not exist in the using period of each product for consumers.

In a first aspect, the present application provides an antibody or antigen-binding fragment thereof that is capable of specifically binding to egg proteins of an ancient elephant pest egg.

As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by a disulfide bond, and multimers thereof (e.g., igM). Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region (abbreviated CH). The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region (abbreviated CL). The light chain constant region comprises one domain (CL 1). The VH and VL regions can be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), into which conserved regions, termed Framework Regions (FRs), are interspersed. In some embodiments, both the light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminus to C-terminus.

As used herein, the term "antigen-binding fragment" of an antibody refers to a portion or segment of an intact antibody molecule that is responsible for binding to an antigen. The antigen-binding fragment may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Antigen-binding fragments of antibodies can be prepared from intact antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, among others. Non-limiting examples of antigen-binding fragments include: fab fragment, fab 'fragment, F (ab') ₂ Fragments, fd fragments, fv fragments, single chain Fv (scFv) molecules, diabodies, dAb fragments, and minimal recognition units (e.g., isolated CDRs) consisting of amino acid residues that mimic hypervariable regions of an antibody. The term "antigen-binding fragment" also includes other engineered molecules, such as diabodies, triabodies, tetrabodies, minibodies, and the like. For example, the Fd fragment herein refers to an antibody fragment consisting of VH and CH1 domains; the Fv fragment consists of the VL and VH domains in a single arm of the antibody; the dAb fragment (Ward et al, nature 1989, 544-546) consists of a VH domain.

An "Fab fragment" comprises one light chain and one heavy chain of CH1 and variable regions. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

A "Fab ' fragment" comprises a light chain and a portion or fragment of a heavy chain comprising the VH and CH1 domains and the region between the CH1 and CH2 domains such that an interchain disulfide bond can form between the two heavy chains of 2 Fab ' fragments to form a F (ab ') 2 molecule.

An "F (ab') 2 fragment" contains two light chains and two heavy chains, which contain a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. The F (ab ') 2 fragment thus consists of two Fab' fragments which are linked together by a disulfide bond between the two heavy chains.

An "Fv fragment" comprises the variable regions from the heavy and light chains, but lacks the constant regions.

"Single chain Fv" or "scFv" refers to antibody fragments comprising the VH domain and the VL domain of an antibody, wherein these domains are present as a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain that enables the scFv to form the desired structure for antigen binding.

"diabodies" refer to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) (VH-VL or VL-VH) in the same polypeptide chain. By using linkers that are too short to allow pairing between two domains on the same chain, each domain is forced to pair with the complementary domain of the other chain, thereby creating two antigen binding sites.

It is well known to those skilled in the art that the complementarity determining regions (CDRs, usually CDR1, CDR2 and CDR 3) are the regions of the variable region that have the greatest impact on the affinity and specificity of an antibody. There are two common definitions of CDR Sequences for VH or VL, namely Kabat definition and Chothia definition, see, for example, kabat et al, "Sequences of Proteins of Immunological Interest", national Institutes of Health, bethesda, MD. (1991); al-Lazikani et Al, J Mol Biol 273 927-948 (1997); and Martin et al, proc.natl.acad.sci.usa 86. For a given antibody variable region sequence, can according to Kabat definition or Chothia definition to determine VH and VL sequence in CDR region sequence. In embodiments of the present application, the CDR sequences are defined using Kabat. Herein, CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively; CDR1, CDR2 and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2 and LCDR3, respectively.

The CDR region sequences in the variable region sequences can be analyzed in a variety of ways for the variable region sequences of a given antibody, such as can be determined using the online software Abysis (http:// www.abysis.org /).

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, e.g., binding of an antibody to an epitope of an antigen, e.g., the ability of an antibody to bind to a specific antigen with an affinity that is at least two times greater than its affinity for a non-specific antigen. It will be appreciated, however, that an antibody is capable of specifically binding to two or more antigens associated with its sequence. For example, an antibody of the present application can specifically bind to B7H3 in humans and non-humans (e.g., mice or non-human primates).

Elephants (Curculionidae) and elephants in the genus archaeus are grain storage pests distributed worldwide. The corn weevil is the biggest harm. Corn elephants are distributed in various provinces of China, are the most main pests of grain foods, are various foods such as rice, corn, sorghum, wheat, peanuts, beans and products and dried fruits thereof, are damaged by adults, are the most seriously damaged by the corn, the wheat and brown rice, and are only damaged by larvae in grains. Broken particles and scraps in the damaged food are increased, the humidity is increased, mites and mould are propagated, the food is mildewed and deteriorated, and the damage is larger. Elephant (s.oryzae) is mainly harmful wheat, barley, oat, rice, corn, sorghum, etc. Rice weevils (s. Granaria) are mainly harmful to various cereals and their processed products, beans, oil, dried fruits, etc.

As used herein, the term "egg protein" refers to a generic term for proteins contained in eggs of Elephantopus pests, and primarily to egg yolk proteins, such as egg yolk proteins having a size of about 170kD in eggs of Oichthys maydis.

In some embodiments, the antibody is selected from the group consisting of a polyclonal antibody and a monoclonal antibody.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations in a small number of individuals.

As used herein, the term "polyclonal antibody" refers to a combination of monoclonal antibodies produced by stimulating the body with multiple epitopes, i.e., polyclonal antibodies. The term "polyclonal antibody" is abbreviated as polyclonal antibody, and its monoclonal antibody corresponds to polyclonal antibody.

In some embodiments, the antibody is encoded by a accession number of CGMCC NO:19184. the hybridoma cell of (3).

In a second aspect, the present application provides a monoclonal antibody represented by the accession number CGMCC NO:19184.

In a third aspect, the present application provides a hybridoma cell having a accession number of CGMCC NO:19184.

in a fourth aspect, the present application provides a test strip for detecting eggs of an ancient elephant pest, comprising the antibody of the first aspect or the monoclonal antibody of the second aspect.

In some embodiments, the test strip can achieve a sensitivity of at least about 0.1%.

In some embodiments, the archaeal pest is selected from the group consisting of hordeolum zearales, archaeological weevils, and hordeolum micranthum.

The term "about" refers to ± 10% of the recited number, for example about 1% refers to a range of 0.9% to 1.1%.

In some embodiments, the test strip can have a sensitivity of at least about 1%, at least about 0.5%, at least about 0.45%, at least about 0.4%, at least about 0.35%, at least about 0.3%, at least about 0.25%, at least about 0.2%, at least about 0.1%, at least about 0.15%, at least about 0.05%, at least about 0.04%, at least about 0.03%, at least about 0.02%, at least about 0.01%, including any whole or fractional number between the above percentages.

In a fifth aspect, the present application provides a kit for detecting eggs of an ancient elephant pest, comprising the antibody of the first aspect, the monoclonal antibody of the second aspect, or the test strip of the fourth aspect.

In a sixth aspect, the application provides a method for detecting eggs of ancient elephant pests, which comprises contacting the antibody of the first aspect, the monoclonal antibody of the second aspect, the reagent of the test strip of the fourth aspect or the kit of the fifth aspect with a sample to be detected,

if the antibody binds to the sample, it indicates the presence of eggs of the archaeological pest in the sample.

In some embodiments, the archaeal pest is selected from the group consisting of hordeolum zearales, archaeological weevils, and hordeolum micranthum.

In some embodiments, the sample to be tested is selected from rice, corn, sorghum, wheat, peanut, legumes, and other crops subject to attack by an ancient elephant pest.

In some embodiments, the test strip cannot be used to detect the presence of other stored grain pests, including but not limited to eggs of tribolium castaneum, sessile beetle, hemimung bean, pisiform, fava bean, coffee bean, indian meal moth, wheat moth, and the like.

In a seventh aspect, the application provides use of the antibody of the first aspect, the monoclonal antibody of the second aspect, the test strip of the fourth aspect, or the kit of the fifth aspect, for detecting the presence of eggs of an archaeological pest in a grain.

In some embodiments, the archaeal pest is selected from the group consisting of hordeolum zearales, archaeological weevils, and hordeolum micranthum.

In some embodiments, the cereal is selected from rice, corn, sorghum, wheat, peanuts, legumes and other crops that are subject to attack by ancient elephant pests.

Examples

The following examples are provided merely to illustrate some embodiments of the present application and are not intended to be limiting in any way. In addition, the methods in the examples will be performed according to conventional protocols in the art, unless otherwise specified.

Example 1 preliminary analysis of egg protein of Ixeris zeae

In the embodiment, the eggs of the elephant corn are obtained by culturing adult elephants corn, and the protein in the eggs is extracted and separated by SDA-PAGE to obtain the target protein.

In brief, fresh rice is taken, adult Zea mays (small amount of adult is taken from natural environment, collected after identifying basic morphological characteristics by stereomicroscope, and cultured continuously in laboratory for propagation) is put into the rice, cultured for 3-7 days at 28 ℃ and humidity of 75 +/-5%, and cultured to obtain rice containing eggs of the Zea mays. Rice was stained with 1% potassium permanganate solution. After dyeing, the rice containing the eggs can be seen to have egg spots left when the corn weevils lay eggs, a certain amount of rice is repeatedly sampled for 3 times, the rice containing the egg spots is counted, and the egg laying rate of the corn weevils cultured at this time is manually calculated. Then, by dissection, the worm eggs in the rice were removed and collected. 2 eggs were ground in 20. Mu.L of lysis buffer (250 mM Tris-HCl (pH 6.8), 10% (W/V) SDS,0.5% (W/V) bromophenol blue, 50% (V/V) glycerol, 5% (W/V) beta-mercaptoethanol). The ground lysate was placed in a boiling water bath for 10 minutes, centrifuged at 10,000rpm (rpm) for 5 minutes, and the supernatant was removed, followed by SDS-PAGE electrophoresis. The control sample was rice without egg.

Referring to fig. 1, lane 1 shows a protein sample of 2 worm eggs, and lanes 2 and 3 show rice protein samples with different concentrations, respectively. As can be seen from the electrophoresis result, the protein with the highest protein abundance in the worm eggs is 170KD and belongs to the protein with large molecular weight. Through literature comparison, the most abundant egg yolk protein in most insect eggs is known, the molecular weight is more than 100kD, and the electrophoresis result of the rice serving as a basic raw material shows that the egg yolk protein basically has no macromolecular protein more than 100 kD. Thereby collecting a large amount of 170KD protein as antigen protein for later antibody preparation.

Example 2 preparation of hybridoma cell lines and monoclonal antibodies thereto

Antigen preparation

Immunogen preparation-hapten is coupled with Bovine Serum Albumin (BSA) to obtain immunogen.

10mg of the hapten (i.e., egg yolk protein) recovered in example 1 was dissolved in 1ml of Dimethylformamide (DMF); taking 15mg of carbodiimide (EDC), fully dissolving the 15mg of carbodiimide (EDC) by using 0.2ml of water, adding the dissolved EDC into a hapten solution, and stirring the mixture for 24 hours at room temperature to obtain a reaction solution A; weighing 30mg of BSA, fully dissolving in 3ml of 0.1mol/L CB (pH 9.6), dropwise and slowly adding the reaction solution A into the protein solution, stirring at room temperature for 24 hours, dialyzing with 0.01mol/L PBS at 4 ℃ for 3 days, and changing the dialysate for 3 times every day to remove unreacted small molecular substances; subpackaging and storing at-20 deg.C for use.

Preparation of coating antigen-coupling hapten and Ovalbumin (OVA) to obtain coating antigen (coating antigen is used for coating on T line of test paper)

10mg of the hapten (i.e. egg yolk protein) recovered in example 1 was taken and dissolved in 1ml of DMF; dissolving 15mg of EDC in 0.2ml of water, adding the solution into the hapten solution, and stirring the solution for 24 hours at room temperature to obtain reaction liquid A; weighing 30mg of OVA, fully dissolving the OVA in 3ml of 0.1mol/L CB (pH 9.6), dropwise and slowly adding the reaction solution A into the protein solution, stirring at room temperature for 24 hours, dialyzing with 0.01mol/L PBS at 4 ℃ for 3 days, and changing the dialysate for 3 times every day to remove unreacted small molecular substances; subpackaging and storing at-20 deg.C for use.

Immunization of animals

The immunogen obtained in the above step is injected into Balb/c mice at an immunization dose of 150. Mu.g/mouse, so that antiserum is produced.

Cell fusion

Cell fusion and cloning: selecting mice with higher serum determination result, taking splenocytes of the mice, fusing the splenocytes with SP2/0 myeloma cells according to the proportion of 8:1 (number ratio), determining cell supernatants by adopting indirect competitive ELISA, and screening positive holes. Cloning the positive hole by using a limiting dilution method until obtaining a hybridoma cell strain secreting the monoclonal antibody.

Freezing and recovering cells: preparation of monoclonal hybridoma cell line into 1 × 10 frozen stock solution ⁶ Cell suspension per ml, preserved for long periods in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for quick melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.

Antibody purification and Performance testing

Production and purification of monoclonal antibodies: injecting 0.5ml of sterilized paraffin oil into Balb/c mice via abdominal cavity, injecting stable monoclonal hybridoma cell strain 5 × 10 after 7 days ⁵ Ascites were collected 7 days later. Purifying ascites by octanoic acid-saturated ammonium sulfate method, and storing at-20 deg.C.

Antibody titer determination (ELISA data): see Table 1 for specific results

TABLE 1

The cell line numbered 1001 obtained in this example was designated WMAZC2 and was subsequently deposited.

And (3) sending WMAZC2 to the common microorganism center of China Committee for culture Collection of microorganisms in 2019, 11 and 28 months, wherein the preservation information is as follows:

the preservation unit is as follows: china general microbiological culture Collection center

The address of the depository: xilu No. 1 Hospital No. 3 of Beijing market Chaoyang district

The preservation date is as follows: 11/28/2019

Culture name (taxonomic nomenclature): monoclonal antibody hybridoma cell of elephant corn worm egg

The preservation number is as follows: CGMCC NO:19184.

example 3 preparation of test strip for detecting egg protein of weevil corn and sample detection

Preparation of colloidal gold

Diluting 1% chloroauric acid to 0.01% (mass fraction) with double distilled deionized water, placing 100ml in a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 2.5ml 1% trisodium citrate under continuous stirring at high temperature, stirring and heating at constant speed until the solution is bright red, cooling to room temperature, recovering the volume with deionized water, and storing at 4 deg.C. The prepared colloidal gold has pure appearance, transparency and no precipitate or floating material.

Preparation of antibody-colloidal gold marker for resisting egg protein of elephant corn

Adjusting pH of the colloidal gold to 7.0 with 0.2mol/L potassium carbonate solution under magnetic stirring, adding antibody against egg protein of Erythrophorus zearales into the colloidal gold solution according to the standard of adding 20-50 μ g per ml of the colloidal gold solution, stirring for 30min, adding 10% BSA to make the final concentration of the solution in the colloidal gold solution 1% (volume fraction), and standing for 10min. Centrifuging at 12000r/min at 4 ℃ for 40min, discarding the supernatant, washing the precipitate twice with a redissolving buffer (0.02-0.1% (mass fraction) containing casein, 0.05-0.2% (mass fraction) Tween-80, and 0.02mol/L phosphate buffer with pH of 7.2), resuspending the precipitate with a redissolving buffer with a volume of 1/10 of the initial volume of colloidal gold, and standing at 4 ℃ for later use.

Preparation of conjugate Release pad

The conjugate release pad was soaked in 0.5mol/L phosphate buffer containing bovine serum albumin (concentration of bovine serum albumin in buffer is 0.5%), pH7.2, and then soaked in lh uniformly, and then baked at 37 ℃ for 3 hrs for use. The prepared antibody-colloidal gold marker for resisting the egg protein of the corn weevil is evenly sprayed on the conjugate release pad by using an Isoflow film spraying instrument, 0.01ml of the antibody-colloidal gold marker for resisting the egg protein of the corn weevil is sprayed on every 1cm of the conjugate release pad, and then the conjugate is placed in an environment with 37 ℃ (the humidity is less than 20%) for 60min, taken out and placed in a dry environment (the humidity is less than 20%) for storage for later use.

Preparation of the reaction film

The coating antigen in example 2 was coated on the reaction membrane to form a detection line (T line), and the goat anti-mouse anti-antibody was coated on the reaction membrane to form a quality control line (C line).

Coating process: diluting the coating source to 10mg/ml by using a phosphate buffer solution, coating the coating source on a detection line on a nitrocellulose membrane by using an Isoflow point membrane instrument, wherein the coating amount is 0.8 mu l/cm; the goat anti-mouse anti-antibody was diluted to 200. Mu.g/ml with 0.01mol/L phosphate buffer pH7.4 and coated on a quality control line on a nitrocellulose membrane in an amount of l.0. Mu.l/cm using an Isoflow dot membrane apparatus. And (3) drying the coated reaction membrane for 2 hours at 37 ℃ for later use.

Preparation of sample absorbent pad

The sample absorption pad is soaked in phosphate buffer solution containing 0.5 percent of bovine serum albumin (volume fraction), pH7.2 and 0.1mol/L for 2h, and is baked for 2h at 37 ℃ for standby.

Assembly of test strips

And sequentially adhering the sample absorption pad, the conjugate release pad, the reaction membrane and the water absorption pad on the PVC bottom plate. The binder release pad is covered by the sample absorption pad from about 1/3 area of the starting end, the end of the binder release pad is connected with the starting end of the reaction membrane, the end of the reaction membrane is connected with the starting end of the water absorption pad, the starting end of the sample absorption pad is aligned with the starting end of the PVC base plate, and the end of the water absorption pad is aligned with the end of the PVC base plate. The reaction film is provided with a detection line and a quality control line, and both the detection line and the quality control line are strip-shaped strips which are vertical to the length direction of the test strip. The detection line is located on the side near the end of the conjugate release pad; the control line is located on the side of the distal end away from the conjugate release pad. The test paper strip is cut into small strips with the width of 3mm by a machine, and the small strips are arranged in a special plastic card and can be stored for 12 months at the temperature of 4-30 ℃ (as shown in figure 2).

And aiming at the sample of the test strip, adding a lowest line of experimental verification detection outside the system. Weighing 20 g of rice samples respectively, adding 0, 1 and 5 eggs added back to the rice samples respectively, soaking 20mL of the PBS buffer solution in a mortar for about 10 minutes, grinding the rice samples until no obvious particles exist, sucking the clear liquid on the upper layer by using a suction pipe after the grinding liquid is settled for 15 minutes, and dropwise adding the clear liquid into a sample hole (S) below the test strip until the sample hole is full. And (3) after the solution permeates for 5 minutes, observing whether the quality control line (C line) in the color development area is developed or not, if the development represents that the detection result is effective, and if the development is not effective, the result is ineffective. If the line C is colored, if the line T below is also colored, the fact that the content of the detected worm eggs exceeds the detection line of the test strip is proved, and if the line T is not colored, the fact that the content of the worm eggs in the sample does not exceed the detection line of the test strip is proved. The result shows that the test strip can effectively detect that 20 g of rice (1000 grains) contains 1 insect egg. That is, the sensitivity of the test strip can reach at least 0.1% (as shown in FIG. 2).

Reference documents:

1. li Yue, guo Daolin, etc. international research on the protection of stored materials, grain storage, 2005 (44): 49-54.

2. Li Yuan, xie Lingde, he Yanping. Determination of the contact toxicity of natural pyrethrin against three stored grain pests.

3. Li Xingkui, wait Jin Li, continue encourage application of biotechnology in comprehensive treatment of stored-material pests, grain science and economy 2014 (39): 51-54.

4. Zhao Linjing, qiao Yan, wang Bin, lemon grass essential oil and its application in food industry, food and fermentation industry 2015 (41): 246-252.

5. Wang Jinglei, xiaoyan bin, li Zengkai, etc. research on indoor control effect of celastrol and diatomite on stored grain pests, grain storage, 2014 (43) 5-8.

Claims (10)

1. An antibody or antigen-binding fragment thereof capable of specifically binding to an egg protein of an insect egg of an archaeological (Sitophilus) pest.

2. The antibody of claim 1, wherein the archaeal pest is selected from the group consisting of hordeolum zearals, archaeological weevil, and hordeolum micranthum.

3. The antibody of claim 1, wherein the antibody is selected from the group consisting of a polyclonal antibody and a monoclonal antibody, optionally, the antigen binding fragment is selected from the group consisting of a Fab fragment, a Fab 'fragment, a F (ab') ₂ Fragments, fd fragments, fv fragments, single chain Fv (scFv) molecules, diabodies, and dAb fragments.

4. The antibody of claim 1, wherein the antibody is encoded by a accession number of CGMCC NO:19184.

5. A monoclonal antibody represented by a sequence with a preservation number of CGMCC NO:19184.

6. A hybridoma cell, wherein the preservation number is CGMCC NO:19184.

7. a test strip for detecting eggs of an archaeological (Sitophilus) pest, comprising the antibody of any one of claims 1-4 or the monoclonal antibody of claim 5, optionally with a sensitivity of up to at least about 0.1%, optionally the archaeological pest being selected from the group consisting of Zedoariae, archaeological and rice weevil.

8. A kit for detecting eggs of an archaeological (Sitophilus) pest, comprising the antibody of any one of claims 1-4, the monoclonal antibody of claim 5, or the test strip of claim 7, optionally, the archaeological pest is selected from the group consisting of zeaxanthin, archaerhodopterus, and mibeheaded elephant.

9. A method for detecting eggs of insect pests belonging to the genus Eleophorus (Sitophilus), which comprises contacting the antibody according to any one of claims 1 to 4, the monoclonal antibody according to claim 5, the test strip according to claim 7 or the reagent in the kit according to claim 8 with a sample to be detected,

if the antibody binds to the sample, indicating the presence of eggs of the archaeological pest in the sample,

optionally, the ancient elephant pest is selected from the group consisting of corn elephant, ancient elephant, and rice elephant,

optionally, the sample to be detected is selected from rice, corn, sorghum, wheat, peanut and beans.

10. Use of the antibody of any one of claims 1 to 4, the monoclonal antibody of claim 5, the test strip of claim 7, or the kit of claim 8 for detecting the presence of eggs of an ancient elephant (Sitophilus) pest in grain,

optionally, the ancient elephant pest is selected from the group consisting of corn elephant, ancient elephant, and rice elephant,

optionally, the cereal is selected from rice, corn, sorghum, wheat, peanut, and legumes.

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